G protein-coupled receptors are ubiquitous components of signal transduction pathways, including many neurotransmitter systems. This project is designed to assess the role of polyunsaturated phospholipids in modulating G protein-coupled signal transduction and to elucidate of the mechanism of action of ethanol in these systems. The visual transduction pathway of the retinal rod photoreceptor is being used as a model system. System properties we are studying include: 1. the kinetics and extent of formation of metarhodopsin II (MII), the G protein activating form of rhodopsin; 2. MII/G protein complex formation; 3. the rate of G protein activation; 4. cGMP phosphodiesterase (PDE) activation; and 5. the GTPase activity of the G protein. Both functional measures in the transduction pathway and acyl chain packing properties of the phospholipid bilayer are being investigated. Current studies demonstrate the importance of membrane hydration in modulating both rhodopsin activation and integrated activity in the signaling pathway. MII formation involves the release of some 34 water molecules, a process, which is enhanced by increased osmolality. An important result of these studies is that in the physiological concentration range of ethanol and at the osmolality of human plasma, the effect of ethanol in enhancing MII formation is increased by a factor of about 2.8. In vitro effects observed in the 100 mM to 280 mM ethanol concentration range are usually considered to be outside the physiological range of interest. However, when measurements are made in the presence of an inert osmotic background, the equivalent results may well be observed in the physiological range of ethanol concentration. In contrast, ethanol has little effect of the PDE activity at physiological levels, but exhibits a moderate osmotic effect at high concentrations, which results in PDE inhibition due to reduced water activity. These findings demonstrate the importance of simulating in vivo conditions to the greatest degree possible, when attempting to evaluate ethanol potency in in vitro experiments. In addition, a generalization of these results suggests that ligand binding and drug partitioning experiments may also be altered dramatically when conducted in the presence of a neutral osmolyte. - neurosciences, nutritional disorders, signal transduction